Western Blots
Total proteins were extracted with protein extraction buffer (Agrisera) supplemented with 5 mM DTT and the cocktail of protease inhibitors to prevent protein degradation. 10 µg of proteins were separated on 15% acrylamide SDS-PAGE and transferred to 0.45 -µm nitrocellulose membrane (Amersham 10600003). All proteins apart from WHY1 were detected with rabbit polyclonal primary antibody (Agrisera) and secondary HRP-linked anti-rabbit (1:10000, Agrisera AS09 602).
For immunological detection of WHY1, the antibodies were directed toward the synthetic peptide of recombinant HvWhy1 protein (PRQYDWARKQVF) in rabbits and antibodies were affinity-purified (Generon, UK). The specificity of immunodetection was validated using pre-immune sera.